The double-stranded (ds) RNA-activated protein kinase, PKR, is one of several proteins induced by interferon and plays a pivotal role in the cellular antiviral response. PKR has also been implicated in cellular signaling involved in transformation, differentiation and apoptosis. PKR is synthesized in a latent state. Binding to dsRNA induces autophosphorylation reactions that activate the kinase. The most well characterized substrate of PKR is the eukaryotic initiation factor elF2alpha; phosphorylation of this protein blocks protein synthesis in virally infected cells. The broad objective of this research program is to define the molecular basis of PKR activation. The critical protein-protein and protein- RNA interactions that modulate the activity of PKR are not well understood. Thus, we propose to define the stoichiometries, affinities and free energy couplings for the formation of macromolecular complexes involved in PKR activation using quantitative solution biophysical methods. Specifically, equilibrium and velocity analytical ultracentrifugation will define the association states and hydrodynamic properties of full-length PKR and domain constructs. These measurements will test the hypothesis that PKR exists in a monomer-dimer equilibrium modulated by phosphorylation state and will probe conformational changes and domain interactions associated with activation. The affinity and orientation of PKR binding to activating and nonactivating synthetic RNAs of variable lengths will be characterized using our novel sedimentation equilibrium method along with CD, fluorescence and affinity cleavage measurements. These studies will test our overlapping ligand model for PKR binding to dsRNA and will correlate RNA binding mode with enzymatic activation. We have designed RNA sequences containing base pair mismatches, bulges and internal loops to systematically probe the structural features that distinguish RNA activators from those that fail to activate. These experiments will be extended to include natural viral RNA activators and inhibitors with complex secondary structures. A detailed mechanistic understanding of PKR activation will provide a foundation for the design of therapeutic agents that target PKR for the treatment of viral infections and cancer.